U.S. patent application number 16/450875 was filed with the patent office on 2020-01-02 for degradation inhibitor of hyaluronic acid, comprising rosemary extract and retinol acetate.
This patent application is currently assigned to Shiseido Company, Ltd.. The applicant listed for this patent is Shiseido Company, Ltd.. Invention is credited to Masato IINO, Nobuhiko OCHIAI, Makoto TSUNENAGA.
Application Number | 20200000704 16/450875 |
Document ID | / |
Family ID | 49778422 |
Filed Date | 2020-01-02 |
![](/patent/app/20200000704/US20200000704A1-20200102-D00001.png)
United States Patent
Application |
20200000704 |
Kind Code |
A1 |
IINO; Masato ; et
al. |
January 2, 2020 |
DEGRADATION INHIBITOR OF HYALURONIC ACID, COMPRISING ROSEMARY
EXTRACT AND RETINOL ACETATE
Abstract
The present invention relates to providing a hyaluronidase
inhibitor having a good hyaluronidase inhibiting activity.
Specifically, the present invention provides with a hyaluronidase
inhibitor comprising rosemary extract having a hyaluronidase
inhibitory activity, and retinol acetate, which can enhances the
hyaluronidase inhibitory activity of rosemary extract.
Inventors: |
IINO; Masato; (Yokohama-shi,
JP) ; OCHIAI; Nobuhiko; (Yokohama-shi, JP) ;
TSUNENAGA; Makoto; (Yokohama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shiseido Company, Ltd. |
Tokyo |
|
JP |
|
|
Assignee: |
Shiseido Company, Ltd.
Tokyo
JP
|
Family ID: |
49778422 |
Appl. No.: |
16/450875 |
Filed: |
June 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13822762 |
Mar 13, 2013 |
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PCT/JP12/66593 |
Jun 28, 2012 |
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16450875 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 8/671 20130101;
A61K 8/9789 20170801; A61P 1/02 20180101; A61P 17/10 20180101; A61P
19/08 20180101; A61P 17/16 20180101; A61Q 19/007 20130101; A61P
17/00 20180101; A61P 43/00 20180101; A61P 37/08 20180101; A61P
17/02 20180101; A61K 2800/782 20130101; A61P 29/00 20180101; A61Q
19/08 20130101 |
International
Class: |
A61K 8/97 20060101
A61K008/97; A61Q 19/08 20060101 A61Q019/08; A61K 8/67 20060101
A61K008/67 |
Claims
1.-2. (canceled)
3. A method for alleviating or preventing one or more skin problems
selected from a group consisting of skin aging, wrinkled skin,
rough skin, dry-and-rough skin, acne and atopic dermatitis in a
subject who is suffering from reduction in hyaluronic acid,
comprising administering a composition comprising between 0.0001
and 0.1 w/w % crude rosemary extract and between 0.000001 and 0.01
w/w % retinol acetate to the subject, wherein the rosemary extract
is extracted from whole plant or leaves of rosemary with
1,3-butylene-glycol aqueous solution.
4. A method of cosmetic treatment comprising applying a composition
comprising between 0.0001 and 0.1 w/w % crude rosemary extract and
between 0.000001 and 0.01 w/w % retinol acetate to a subject
suffering from a reduction in hyaluronic acid, wherein the rosemary
extract is extracted from whole plant or leaves of rosemary with
1,3-butylene-glycol aqueous solution.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 13/822,762, which is the National Stage application of
PCT/JP2012/066593, filed Jun. 28, 2012.
TECHNICAL FIELD
[0002] The present invention relates to an inhibitor for
hyaluronidase which has a good hyaluronidase inhibitory
activity.
BACKGROUND ART
[0003] Recently, research studies relating to aging have been
conducted. In a macroscopic aspect, increased age is a major cause
for skin aging. In addition to increased age, the influences
resulted from drying, oxidizing, and sunlight (ultraviolet rays)
were also known as a direct cause for skin aging. A reduction of
mucopolysaccharides, such as hyaluronic acid, etc., cross-linking
reaction of collagen, and cellular damage due to ultraviolet rays
were known as a concrete phenomenon of skin aging.
[0004] Inter alia, hyaluronic acid, one of glycosaminoglycans,
which is widely present in the connective tissue of mammals along
with chondroitin sulfate, etc., and is a linear high molecular
polysaccharide comprising .beta.-D-N-acetylglucosamine and
.beta.-D-glucuronic acid alternately bound to each other, has
various functions, such as retaining water in the intercellular
space, holding cells by forming a gelatinous matrix in the tissue,
maintaining lubricity and flexibility of the tissue, resisting
against external force applied, such as mechanical stress, and
inhibiting cellular infection (See, NPL 1). In addition, the amount
of hyaluronic acid is decreased with increasing age, thereby skin
aging, such as a finely wrinkled skin or dry and rough skin is
thought to appear. Therefore, many cosmetics comprising hyaluronic
acid are provided as an aged skin improving agent. However, such
previous cosmetics cannot improve the aged skin fundamentally, but
merely exert a moisture retention effect on the skin surface.
Although several cosmetics comprising an agent that increases the
amount of hyaluronic acid in the skin have been recently provided
(see, PLT1), such cosmetics are not a product which can clearly
improve and treat the aged skin. It was believed that the aged skin
cannot necessarily be improved by externally applying hyaluronic
acid or an agent which accelerates production of hyaluronic acid to
the skin, since hyaluronic acid degrades promptly in the skin. Once
dermal hyaluronic acid is synthesized in vivo, it is immediately
subjected to the enzymatic degradation from hyaluronidase, a
degradative enzyme. Therefore, the degradation rate is high, and
the half-life of hyaluronic acid is reported to be about one day
(see, NRP2). Hyaluronidase is an enzyme which can degrade
hyaluronic acid as speculated from its name, and is distributed
widely in the living body and also present in the skin. Therefore,
it was believed that the inhibition of the activity of
hyaluronidase, which promotes the degradation of hyaluronic acid,
can contribute to the stability of hyaluronic acid used in a
formulation, and also to the stability of hyaluronic acid
originally present in the skin or derived from the formulation
after applying it to the skin. In addition, since hyaluronidase was
also known as an inflammatory enzyme, inhibiting the activity
thereof can inhibit inflammation, and can also repress allergy. Due
to the above reasons, in order to maintain skin elasticity and
moisture retaining property, thereby preventing wrinkled, finely
wrinkled and dry-and-rough skin and maintaining a moist and fresh
skin condition, the development of an effective inhibitor for
hyaluronidase, a hyaluronic acid degrading enzyme, has been
required.
[0005] Extract of a plant of the Labiatae family, such as rosemary,
thyme, and melissa, etc., has been shown to inhibit hyaluronidase,
suppress platelet aggregation, and suppress exosaminidase
liberation, and therefore the extract are shown to have an
anti-inflammatory effect (PLT2). A hyaluronidase inhibitory
substance extracted from any one of rosemary, thyme, or melissa, a
mixture thereof, and a mixture further comprising an anti-allergic
agent were described in the document. However, there was no
description relating to a mixture which can exert a synergic effect
over an additive effect.
CITATION LIST
Patent Literature
[0006] PLT1: Japanese Unexamined Patent Publication (Kokai) No.
11-209261 [0007] PLT2: Japanese Unexamined Patent Publication
(Kokai) No. H08-333267
Non Patent Literature
[0007] [0008] NPL1: "BioIndustry", vol. 8, p. 346 (1991) [0009]
NPL2: Tammi R. et al., J. Invest. Dermatol., 97, 126-130 (1991)
SUMMARY OF INVENTION
Technical Problem
[0010] The present inventions are premised on the above
circumstance, and are intended to provide a hyaluronidase inhibitor
having a hyaluronidase inhibitory activity in the human body which
is safe and can be easily used.
Solution to Problem
[0011] In order to solve the above problem, the inventors have
carried out extensive researches for screening widely various
materials on a hyaluronidase inhibitory activity, and surprisingly
found that a hyaluronidase inhibitory activity is drastically
enhanced when rosemary extract, which is known to exert a
hyaluronidase inhibitory activity, is used in combination with
Vitamin A, which is not known to exert a hyaluronidase inhibitory
activity. Based on this discovery, the inventors have completed the
present invention.
[0012] Accordingly, the present invention is directed to the
following inventions:
[1] A hyaluronidase inhibitor comprising rosemary extract and
vitamin A. [2] The hyaluronidase inhibitor of [1], wherein the
vitamin A is selected from the group consisting of retinol, retinol
acetate, retinol palmitate. [3] The hyaluronidase inhibitor of [1]
or [2], wherein the vitamin A is retinol acetate. [4] A skin
external agent for alleviating or preventing skin problems selected
from the group consisting of skin aging, wrinkled skin, rough skin,
dry skin, rough-and-dry skin, acne and atopic dermatitis,
comprising the hyaluronidase inhibitor of any one of [1]-[3]. [5]
An external skin agent for treating a disease related to a
reduction in hyaluronic acid selected from the group consisting of
atopic dermatitis, dry skin, arthritis, gingivitis, rheumatism,
osteoarthritis, and burn injury, comprising the hyaluronidase
inhibitor of any one of [1]-[3]. [6] Use of rosemary extract and
vitamin A for improving or preventing a reduction in hyaluronic
acid. [7] Use of rosemary and vitamin A for treating or preventing
skin problems, such as skin aging, wrinkled skin, dry skin, dry and
rough skin, acne, and atopic dermatitis, or for treating a disease
related to a reduction in hyaluronic acid, such as dry skin,
arthritis, ingivitis, rheumatism, osteoarthritis, and burn injury.
[8] A method for inhibiting hyaluronidase, comprising administering
rosemary extract and vitamin A to a subject suffering from a
reduction in hyaluronic acid. [9] A method of cosmetic or medical
treatment for alleviating or preventing a reduction in hyaluronic
acid, comprising administering rosemary and vitamin A to a subject
suffering from a reduction in hyaluronic acid. [10] The method of
[9], wherein the method of cosmetic or medical treatment is
intended for improving or preventing skin problems, such as
wrinkled skin, rough skin, dry skin, dry-and-rough skin, acne, and
atopic dermatitis. [11] A method for treating a disease related to
a reduction in hyaluronic acid, such as atopic dermatitis, dry
skin, arthritis, gingivitis, rheumatism, osteoarthritis, and burn
injury. [12] Use of rosemary extract and vitamin A, for preparing a
hyaluronidase inhibitor. [13] Use of rosemary extract and vitamin A
for preparing cosmetic or pharmaceutical product for improving or
preventing skin problems, such as skin aging, wrinkled skin, rough
skin, dry skin, dry-and-rough skin, acne, and atopic dermatitis.
[14] Use of rosemary extract and vitamin A, for preparing a
pharmaceutical product for treating a disease related to a
reduction in hyaluronic acid, such as atopic dermatitis, dry skin,
arthritis, gingivitis, rheumatism, osteoarthritis, and burn
injury.
Advantageous Effects of Invention
[0013] When vitamin A, which is included in a hyaluronidase
inhibitor of the present invention, is used alone, a hyaluronidase
inhibitory activity is not exerted, or is hardly exerted. However,
when vitamin A is used in combination with rosemary extract,
vitamin A significantly enhances the hyaluronidase inhibitory
activity of rosemary extract. Therefore, the hyaluronidase
inhibitor of the present invention exerts either effects of
improving or preventing skin problems, such as human skin aging,
wrinkled skin, rough skin, dry skin, dry-and-rough skin, acne, and
atopic dermatitis, or improving or preventing disease related to a
reduction in hyaluronic acid, such as arthritis, gingivitis,
rheumatism, osteoarthritis, or burn injury, etc.
BRIEF DESCRIPTION OF DRAWINGS
[0014] FIG. 1 shows that while rosemary extract has a hyaluronidase
activity, but retinol acetate does not have a hyaluronidase
activity, the hyaluronidase inhibitory activity of rosemary extract
can be significantly enhanced by combined use of rosemary extract
and retinol acetate.
DESCRIPTION OF EMBODIMENTS
[0015] In the present invention, hyaluronidase is a collective term
of an enzyme which degrades hyaluronic acid by cleaving a glucoside
linkage. Since hyaluronidase is widely distributed in the mammal
tissue, the amount of hyaluronic acid inside the body, in
particular inside the skin or joint fluid, is expected to be
increased by inhibiting the activity of hyaluronidase. Glycoside
linkage to be cleaved can be glucronide linkage or
N-acetylglucosaminide linkage.
[0016] In the present invention, a hyaluronidase inhibitor is an
agent inhibiting a hyaluronic acid degradation activity of
hyaluronidase, and therefore it can increase the amount of
hyaluronic acid inside the body, in particular joint fluid or skin.
In a preferable aspect of the present invention, a hyaluronidase
inhibitor includes rosemary extract and vitamin A.
[0017] Hyaluronic acid relates to elasticity, moisture retaining
property, and lubricity inside the body. It is also known that the
amount of hyaluronic acid diminishes with aging, and that symptoms,
such as wrinkled skin, rough skin, dry skin or dry-and-rough skin
appear due to a reduction in hyaluronic acid in the skin. In
addition, it is also known that hyaluronidase relates to
inflammatory, and that the activity of hyaluronidase relates to
atopic dermatitis or acne. Therefore, the skin problems, such as
aging, wrinkled skin, rough skin, dry skin, dry-and-rough skin,
acne, or atopic dermatitis, and diseases related to a reduction in
hyaluronic acid, such as dry skin, arthritis, gingivitis,
rheumatism, osteoarthritis, and burn injury can be treated,
improved or prevented by the hyaluronidase inhibitor of the present
invention.
[0018] In the present invention, rosemary extract means an extract
derived from Lamiaceae (family), Rosmarinus (genus) rosemary
(Rosmarinus officinalis), which is indigenous to the Mediterranean
region. All parts of rosemary, in particular the whole plant, leaf,
and flower can be used as a source material. Rosemary extract is
known to have an antibiotic property, antioxidant property, and
anti-inflammatory property, and therefore it was reported that
rosemary extract can be used as a pharmaceutical product, such as
an anti-aging agent, such as a skin improvement agent; an
immunomodulatory agent; an anti-diabetic agent; an
anti-osteoporotic agent; an anti-adiposity agent; a sleep enhancing
agent; anti-central nervous agent; etc.
[0019] The rosemary extract of the present invention can be
prepared in accordance with a known method, for example by
immersing the plant in extracting solvent, or heating to reflux the
solvent, and then carrying out a filtration and condensation. Any
extracting solvents usually used in an extraction can be used. For
example, water, alcohols, such as methanol, ethanol, propylene
glycol, 1,3-buthyleneglycol, glycerine, hydroalcoholic solutions,
chloroform, dichlorethane, carbon tetrachloride, acetone, ethyl
acetate, hexane, etc., can be used independently or in combination
as a extracting solvent. An extract obtained by extracting with the
above solvent can be used without purification. Alternatively, an
extract which is subjected to an adsorption method using, for
example, an ionic exchange resin, so as to remove impurities, or
which is subjected to a porous polymer column (for example
Amberlite XAD-2) to adsorb thereon, eluted with methanol or
ethanol, and is then concentrated, can be used. A dried extract can
also be used. In addition, an extract which is extracted according
to a distribution method, for example by using water/ethyl acetate,
can also be used.
[0020] The extract of the plant obtained thereby has a good
hyaluronidase inhibitory activity. Since the extract is used as a
cosmetic or pharmaceutical product, which is directly applied to
the skin, such as a skin external agent, etc., the extract
extracted with water, 1,3-butylene glycol, glycerine, etc., is
preferable. For example, the extract extracted prepared by means of
50 w/w % 1,3-butyleneglycol aqueous solution is more
preferable.
[0021] When the above plant or its extract is contained in a
cosmetic or pharmaceutical product, it is contained in the cosmetic
or pharmaceutical product at 0.00001 to 0.1 w/w %, preferably
0.00005 to 0.01 w/w %, more preferably 0.0001 to 0.001 w/w %, at
dry weight.
[0022] A deficiency of Vitamin A is known to induce night blindness
or rachitis, and relating to the skin, also known to result in
keratinization of skin or mucosa, abnormal dryness of skin, and
pigment deposition. Therefore, vitamin A has been used for skin in
order to treat acne or promote skin turn over. However, it is not
described nor suggested that vitamin A enhances a hyaluronidase
inhibitory activity. Vitamin A includes one or more ingredients
selected from vitamin A group, such as retinol, retinal, retinoic
acid, and a precursor or derivative thereof, such as retinol
acetate, retinol palmitate, tretionic acid, methyl retinate, ethyl
retinate, retinoic acid retinol. In view of applying it to skin,
retinol, retinol acetate, or retinol palmitate is preferable, and
retinol acetate is most preferable.
[0023] The amount of vitamin A used in the present invention is not
limited as long as a hyaluronidase inhibitory effect of rosemary
extract can be enhanced. For example, vitamin A is usually included
in a cosmetic or pharmaceutical product at 0.000001-0.01 w/w %,
preferably 0.000005-0.001 w/w %, more preferably 0.00001-0.0001 w/w
% in total.
[0024] Any mixing rate of rosemary extract and vitamin A can be
used as long as a hyaluronidase inhibitory effect of rosemary
extract can be enhanced. For example, 0.01 weight parts-5 weight
parts, preferably 0.05 weight parts-1 weight parts, more preferably
0.1 weight parts-0.5 weight parts of vitamin A can be used per 1
weight part of rosemary extract.
[0025] Since vitamin A does not usually have a hyaluronidase
inhibitory activity, it can be said that a synergistic effect
surpassing an additive effect is exerted by using vitamin A in
combination with rosemary extract, if vitamin A could enhance the
hyaluronidase inhibitory activity of rosemary extract. Even if
vitamin A has a hyaluronidase inhibitory activity, the activity is
weak, whereas vitamin A highly enhances the hyaluronidase
inhibitory activity of rosemary extract when used in combination
with rosemary extract. Therefore, even in such a case, a
synergistic effect surpassing an additive effect is exerted by the
combination of vitamin A and rosemary extract.
[0026] The hyaluronidase inhibitor of the present invention can be
further contained in a cosmetic or pharmaceutical product.
Therefore, in addition to rosemary extract and vitamin A, if
necessary, an active ingredient and/or excipient, for example, a
moisturizing agent, an antioxidant, an oily material, an UV
protective agent, a surfactant, a thickening agent, a base
ingredient, such as alcohols, glycerine, hyalronic acid, a pH
modifier powder ingredient, an UV absorbing agent, a stabilizing
agent, an antimicrobial agent, a flavoring agent, a colorant agent,
water, several skin nutrition agents, etc., can be arbitrarily
included in the cosmetic or pharmaceutical product, as long as the
effect of the present invention is not impaired.
[0027] In addition, a sequestering agent, such as disodium edetate,
trisodium edetate, sodium citrate, sodium polyphosphate, sodium
metaphosphate, gluconic acid, etc., an antiseptic agent, such as
methyl paraben, ethyl paraben, buthyl paraben, etc., a drug, such
as caffeine, tannin, verapamil, tranexamic acid and a derivative
thereof, licorice extract, glabridin, quince fruit hot water
extract, various crude drugs, tocopherol acetate, glycyrrhizic acid
and a derivative or salt thereof, a whitening agent, such as
vitamin C, magnesium ascorbyl phosphate, ascorbic acid Glucoside,
arbutin, Kojic acid, etc., saccharides, such as glucose, fructose,
mannose, sucrose, treharose, etc., can also be arbitrarily
included.
[0028] In addition, a cosmetic or pharmaceutical product comprising
the hyaluronidase inhibitor of the present invention can be a skin
external agent, such as a cosmetic product, a quasi-pharmaceutical
product, etc., which is applied externally to the skin. Any
formulations, such as solution system, a solubilized system, a
emulsified system, a powder dispersal system, a water-oil bilayer
system, a water-oil-powder trilayer system, an ointment, skin
lotion, gel, aerozol, etc., can be applied.
[0029] A cosmetic product comprising the hyaluronidase inhibitor of
the present invention includes, but is not limited to, a facial
cosmetic product, such as skin lotion, milky lotion, cream, beauty
serum, facial mask, a makeup cosmetic product, such as foundation,
lip rouge, eye shadow, a sun protective agent, a perfumed cosmetic
product, a hair cosmetic product, or a bath agent.
[0030] In addition to transdermal administration, the hyaluronidase
inhibitor of the present invention can be administered orally, or
parentally (for example, transmucossally, sublingually,
intramuscularly, intra-articularly or subcutaneously). Therefore,
in addition to a cosmetic or pharmaceutical product used for skin
external use for preventing skin problems, such as wrinkled skin,
rough skin, dry skin, dry-and-rough skin, acne or atopic
dermatitis, the hyaluronidase inhibitor of the present invention
can be included in food for preventing a reduction in hyaluronic
acid in the body which is caused by aging, and a pharmaceutical
composition, such as an agent for treating hyaluronic acid abnormal
degradation disease, which is used for treating or preventing
symptoms in which hyaluronic acid degradation is abnormally
enhanced, in particular gingivitis, rheumatism, osteoarthritis, or
arthritis, or is used for initial treatment of burn injury. When
the hyaluronidase inhibitor of the present invention is used for a
pharmaceutical composition, the formulation is not limited, but can
be arbitrarily selected depending on an administration route. For
example, a formulation suitable for oral administration includes a
tablet, capsule, powder drug, fine grain drug, granule, liquid
drug, syrup, etc., and a formulation suitable for parental
administration includes an injectable drug, drops, suppository,
inhalation, topical drug, transmucosal drug, adhesive patch, etc.
The injectable drug can be used for any one of intraarticular
injection, intravenous injection, intramuscular injection,
subcutaneous injection, or drop infusion.
[0031] The present invention is directed to a method of cosmetic
treatment or medical treatment for reducing or preventing a
decrease in hyaluronic acid, comprising applying rosemary extract
and vitamin A to a subject suffering from a reduction in hyaluronic
acid. Rosemary extract and vitamin A can be applied at the same
time or separately at intervals. The method of cosmetic treatment
is directed to not only a method which is carried out individually,
but also a method which is provided by a person other than a
physician, such as a sales staff for cosmetic products or a beauty
therapist, wherein the method is provided as a prescription of a
cosmetic product which is suitable for a customer at the time of
providing a cosmetic product.
[0032] In addition to a subject who is suffering from a reduction
in hyaluronic acid with aging, a subject suffering from a reduction
in hyaluronic acid includes a subject who is suffering from a
disease relating to a reduction in hyaluronic acid, such as atopic
dermatitis, dry skin, arthritis, gingivitis, rheumatism,
osteoarthritis, burn injury, etc.
EXAMPLES
[0033] The present inventions are further explained in detail in
the following Examples. However, the technical scope of the present
invention should not be restricted by these examples. In addition,
all percentages mean w/w %.
Preparation Example: Preparation of Rosemary Extract
[0034] 50 w/w % 1,3-butylene glycol aqueous solution was added to
10 kg of cut leaves of rosemary, and then the leaves were immersed
for 7 to 10 days at room temperature. After the immersion,
undesired substances were removed through filtration, and the
filtrate was further incubated for 7 to 10 days in a cold location.
After the incubation, undesired substances were removed through
filtration so as to obtain rosemary extract BG.
Example: An Examination for Inhibiting Hyaluronic Acid
Degradation
[0035] 50 .mu.l hyaluronidase solution (1 mg/ml) (Sigma Aldrich
Japan K.K.) was placed into a microtube, and 50 .mu.l of an
evaluation drug was added, and then the mixture was incubated for
10 min at 37.degree. C. Rosemary extract BG (Maruzen
Pharmaceuticals, Co., Ltd.), retinol acetate (BASF Japan Ltd.), and
rosemary extract and retinol acetate were used as the evaluation
drug, and water was used as a control instead of the evaluation
agent. Rosemary extract BG was added so that the final
concentration thereof after addition of hyaluronic acid sodium
aqueous solution was 0.000165 w/w % at dry weight. Retinol acetate
was added so that the final concentration thereof after addition of
hyaluronic acid sodium aqueous solution was 0.0000516 w/w %. After
incubation, 400 .mu.l of 1 mg/ml sodium hyaluronate (Shiseido Co.,
Ltd., MW 1,100,000-1,600,000) was added to the solution, and then
incubated at 37.degree. C., for 10 min, and then 50 .mu.l of sodium
hydroxide (0.2 M) was added to stop the reaction. This solution was
boiled for 3 min, then cooled with flowing water. 300 .mu.l of 10%
p-DMAB (dimethylaminobenzaldehyde) solution (NACALAI TESQUE, Inc.)
was added to the solution, and then incubated for 37.degree. C. for
20 min. After bringing it back to room temperature, 100 .mu.l of
each solution was poured into each well of 96 well-plate, and OD
585 nm was determined by using POWERSCAN HT (DS Pharma Biomedical
Co., Ltd.). The result wherein the absorbance in a control well was
adjusted to be 100 was shown in FIG. 1. FIG. 1 showed that rosemary
extract had a hyaluronidase inhibitory activity by itself, whereas
retinol acetate did not have a hyaluronidase inhibitory activity,
and that when rosemary extract was used in combination with retinol
acetate, the hyaluronidase inhibitory activity of the rosemary
extract was enhanced.
TABLE-US-00001 (w/w %) Prescription Example (skin lotion) Ethanol
5.0 Glycerine 0.5 Dipropylene glycol 2.0 1,3-butylene glycol 6.0
Sage oil 0.01 Citric acid 0.02 Sodium Citrate 0.08 Sodium
hexametaphosphate 0.03 Hydroxy propyl-.beta.-cyclodextrin 0.1
Retinol acetate 0.00005 Rosemary extract (dry weight) 0.001 Rosa
Multiflora Fruit extract (dry weight) 0.001 Phellodendoron Amurense
Bark extract (dry weight) 0.001 Rosa roxburghii fruit extract (dry
weight) 0.001 Lavender oil 0.1 Prums Persica (Peach) Kernel Extract
(dry weight) 0.001 Sodium arginate 0.001 Purified water residue
Prescription Example (milky lotion) Dimethyl polysiloxane 2.0
Behenyl alcohol 1.0 Batyl Alcohol 0.5 Glycerine 5.0 1,3-butylene
glycol 7.0 Erythritol 2.0 Hardened oil 3.0 Scwaran 6.0 Tetra
2-ethylhexanoic acid pentaerythritol 2.0 Isostearic acid
polyoxyethylene glycerol 1.0 Mono Stearic acid polyoxyethylene
glycerine Retinol acetate 0.0001 Rosemary extract (dry weight)
0.001 Potassium hydroxide ad lib. hexametaphosphoric acid 0.05
Phenoxy ethanol ad lib. Carboxyvinyl polymer 0.1 Purified water
residue Prescription Example (Cream) Liquid paraffin 8.0 Vaseline
3.0 Dimethyl polysiloxane 2.0 Stearyl alcohol 3.0 Behenyl alcoho
2.0 Glycerine 5.0 Dipropylene glycol 4.0 Trehalose 1.0 Tetra
2-ethylhexanoic acid pentaerythritol 4.0 Monoisostearic acid
polyoxyethylene glycerol 2.0 Monostearic acid polyoxyethylene
glycerine 1.0 Lipophilic monostearic acid glycerine 2.0 Citric acid
0.05 Sodium citrate 0.05 Potassium hydroxide 0.015 Oil soluble
licorice extract (dry weight) 8.0 Retinol acetate 0.0001 Tocopherol
acetate 0.1 Rosemary extract (dry weight) 0.0002 Paraoxybenzoic
acid ester ad lib. Phenoxy ethanol ad lib. Dibutyl hydroxy toluene
ad lib. Trisodium edetate 0.05 4-t-butyl-4'-methoxydibenzoylmethan
0.01 Paramethoxy cinnamic acid 2-ethyl hexyl 0.1 .beta.-carotene
0.01 Polyvinyl alcohol 0.5 Hydroxyethylcellulose 0.5 Carboxy vinyl
polymer 0.05 Purified water residue Flavoring agent ad lib.
* * * * *